The biosynthesis of active opioid peptides, like the biosynthesis of other peptides and hormones, involves a sequential series of enzymatic steps begin with signal peptide cleavage and ending with various terminal modifications. Considerable information is available on the processing steps involved in the production of ACTH and beta endorphin from the opioid peptide precursor proopio- melanocortin; however, the processing pathways taken by peptides derived from the other two opioid peptide precursors, proenkephalin and prodynorphin, are not well understood. Since opioid peptides have been implicated in physiological processes ranging from blood pressure control to immunological function, it is important to understand the biosynthetic mechanisms responsible for enkephalin production. We have previously carried out studies on the biosynthesis of enkephalins in the adrenal medulla. We now propose to extend and complete these studies by establishing the sequence of posttranslational processing steps required for the production of the penta- to octapeptide enkephalins met-enkephalin, leu- enkephalin, met-enkephalin-arg-phe, and met-enkephalin-arg-gly- leu. We will rely heavily on the use of pulse- chase/immunoprecipitation techniques to carry out this work. We also propose to follow up our discovery of the phosphorylation of proenkephalin with studies on the regulation of this important posttranslational processing event, and explore the potential presence of other such modifications of enkephalin-derived peptides (such as sulfation and glycosylation). It is hoped that information gained through these studies will be of use in future investigations of opioid peptides within the nervous system, and will provide basis for the study of plasma enkephalins as hormonal regulators.